Cardiomyocyte Orientation Research Articles

Cardiomyocyte orientation recovery at micrometer scale reveals long-axis fiber continuum in heartwalls.

Coordinated cardiomyocyte contraction drives the mammalian heart to beat and circulate blood. No consensus model of cardiomyocyte geometrical arrangement exists, due to the limited spatial resolution of whole heart imaging methods and the piecemeal nature of studies based on histological sections. By combining microscopy and computer vision, we produced the first-ever three-dimensional cardiomyocyte orientation reconstruction across mouse ventricular walls at the micrometer scale, representing a gain of three orders of magnitude in spatial resolution. We recovered a cardiomyocyte arrangement aligned to the long-axis direction of the outer ventricular walls. This cellular network lies in a thin shell and forms a continuum with longitudinally arranged cardiomyocytes in the inner walls, with a complex geometry at the apex. Our reconstruction methods can be applied at fine spatial scales to further understanding of heart wall electrical function and mechanics, and set the stage for the study of micron-scale fiber remodeling in heart disease.

On the structural origin of the anisotropy in the myocardium: Multiscale modeling and analysis

Due to structural heterogeneities within the tissue, the myocardium displays an orthotropic material behavior. However, the link between the microstructure and the macroscopic mechanical properties is still not fully established. In particular, if it is admitted that the cardiomyocyte organization induces a transversely isotropic symmetry, the relative role in the observed orthotropic symmetry of cardiomyocyte orientation variation and perimysium collagen “sheetlet” structure, two mechanisms occurring at different scales, is still a matter of debate. In order to shed light on this question, we designed a multiscale model of the myocardium, bridging the cell, sheetlet and tissue scales. More precisely, we compared the macroscopic anisotropy obtained by homogenization of different mesostructures consisting in cardiomyocytes and extracellular collageneous layers, also taking into account the variation of cardiomyocyte and sheetlet orientations on the macroscale, to available experimental data. This study confirms the importance of sheetlets layers in assuring the tissue’s anisotropic response, as cardiomyocytes-only mesostructures cannot reproduce the observed anisotropy. Moreover, our model shows the existence of a size effect in the myocardial tissue shear properties, which will require further experimental analysis.

Myocardial mesostructure and mesofunction.

The complex and highly organized structural arrangement of some five billion cardiomyocytes directs the coordinated electrical activity and mechanical contraction of the human heart. The characteristic transmural change in cardiomyocyte orientation underlies base-to-apex shortening, circumferential shortening, and left ventricular torsion during contraction. Individual cardiomyocytes shorten ∼15% and increase in diameter ∼8%. Remarkably, however, the left ventricular wall thickens by up to 30-40%. To accommodate this, the myocardium must undergo significant structural rearrangement during contraction. At the mesoscale, collections of cardiomyocytes are organized into sheetlets, and sheetlet shear is the fundamental mechanism of rearrangement that produces wall thickening. Herein, we review the histological and physiological studies of myocardial mesostructure that have established the sheetlet shear model of wall thickening. Recent developments in tissue clearing techniques allow for imaging of whole hearts at the cellular scale, whereas magnetic resonance imaging (MRI) and computed tomography (CT) can image the myocardium at the mesoscale (100 µm to 1 mm) to resolve cardiomyocyte orientation and organization. Through histology, cardiac diffusion tensor imaging (DTI), and other modalities, mesostructural sheetlets have been confirmed in both animal and human hearts. Recent in vivo cardiac DTI methods have measured reorientation of sheetlets during the cardiac cycle. We also examine the role of pathological cardiac remodeling on sheetlet organization and reorientation, and the impact this has on ventricular function and dysfunction. We also review the unresolved mesostructural questions and challenges that may direct future work in the field.

Comparison of interpolation methods of predominant cardiomyocyte orientation from in vivo and ex vivo cardiac diffusion tensor imaging data.

Cardiac electrophysiology and cardiac mechanics both depend on the average cardiomyocyte long‐axis orientation. In the realm of personalized medicine, knowledge of the patient‐specific changes in cardiac microstructure plays a crucial role. Patient‐specific computational modelling has emerged as a tool to better understand disease progression. In vivo cardiac diffusion tensor imaging (cDTI) is a vital tool to non‐destructively measure the average cardiomyocyte long‐axis orientation in the heart. However, cDTI suffers from long scan times, rendering volumetric, high‐resolution acquisitions challenging. Consequently, interpolation techniques are needed to populate bio‐mechanical models with patient‐specific average cardiomyocyte long‐axis orientations. In this work, we compare five interpolation techniques applied to in vivo and ex vivo porcine input data. We compare two tensor interpolation approaches, one rule‐based approximation, and two data‐driven, low‐rank models. We demonstrate the advantage of tensor interpolation techniques, resulting in lower interpolation errors than do low‐rank models and rule‐based methods adapted to cDTI data. In an ex vivo comparison, we study the influence of three imaging parameters that can be traded off against acquisition time: in‐plane resolution, signal to noise ratio, and number of acquired short‐axis imaging slices.

Quantification of Myocyte Disarray in Human Cardiac Tissue.

Proper three-dimensional (3D)-cardiomyocyte orientation is important for an effective tension production in cardiac muscle. Cardiac diseases can cause severe remodeling processes in the heart, such as cellular misalignment, that can affect both the electrical and mechanical functions of the organ. To date, a proven methodology to map and quantify myocytes disarray in massive samples is missing. In this study, we present an experimental pipeline to reconstruct and analyze the 3D cardiomyocyte architecture in massive samples. We employed tissue clearing, staining, and advanced microscopy techniques to detect sarcomeres in relatively large human myocardial strips with micrometric resolution. Z-bands periodicity was exploited in a frequency analysis approach to extract the 3D myofilament orientation, providing an orientation map used to characterize the tissue organization at different spatial scales. As a proof-of-principle, we applied the proposed method to healthy and pathologically remodeled human cardiac tissue strips. Preliminary results suggest the reliability of the method: strips from a healthy donor are characterized by a well-organized tissue, where the local disarray is log-normally distributed and slightly depends on the spatial scale of analysis; on the contrary, pathological strips show pronounced tissue disorganization, characterized by local disarray significantly dependent on the spatial scale of analysis. A virtual sample generator is developed to link this multi-scale disarray analysis with the underlying cellular architecture. This approach allowed us to quantitatively assess tissue organization in terms of 3D myocyte angular dispersion and may pave the way for developing novel predictive models based on structural data at cellular resolution.

Estimating Aggregate Cardiomyocyte Strain Using In Vivo Diffusion and Displacement Encoded MRI.

Changes in left ventricular (LV) aggregate cardiomyocyte orientation and deformation underlie cardiac function and dysfunction. As such, in vivo aggregate cardiomyocyte "myofiber" strain ( [Formula: see text]) has mechanistic significance, but currently there exists no established technique to measure in vivo [Formula: see text]. The objective of this work is to describe and validate a pipeline to compute in vivo [Formula: see text] from magnetic resonance imaging (MRI) data. Our pipeline integrates LV motion from multi-slice Displacement ENcoding with Stimulated Echoes (DENSE) MRI with in vivo LV microstructure from cardiac Diffusion Tensor Imaging (cDTI) data. The proposed pipeline is validated using an analytical deforming heart-like phantom. The phantom is used to evaluate 3D cardiac strains computed from a widely available, open-source DENSE Image Analysis Tool. Phantom evaluation showed that a DENSE MRI signal-to-noise ratio (SNR) ≥20 is required to compute [Formula: see text] with near-zero median strain bias and within a strain tolerance of 0.06. Circumferential and longitudinal strains are also accurately measured under the same SNR requirements, however, radial strain exhibits a median epicardial bias of -0.10 even in noise-free DENSE data. The validated framework is applied to experimental DENSE MRI and cDTI data acquired in eight ( N=8 ) healthy swine. The experimental study demonstrated that [Formula: see text] has decreased transmural variability compared to radial and circumferential strains. The spatial uniformity and mechanistic significance of in vivo [Formula: see text] make it a compelling candidate for characterization and early detection of cardiac dysfunction.

Cardiomyocyte orientation modulated by the Numb family proteins–N-cadherin axis is essential for ventricular wall morphogenesis

The roles of cellular orientation during trabecular and ventricular wall morphogenesis are unknown, and so are the underlying mechanisms that regulate cellular orientation. Myocardial-specific Numb and Numblike double-knockout (MDKO) hearts display a variety of defects, including in cellular orientation, patterns of mitotic spindle orientation, trabeculation, and ventricular compaction. Furthermore, Numb- and Numblike-null cardiomyocytes exhibit cellular behaviors distinct from those of control cells during trabecular morphogenesis based on single-cell lineage tracing. We investigated how Numb regulates cellular orientation and behaviors and determined that N-cadherin levels and membrane localization are reduced in MDKO hearts. To determine how Numb regulates N-cadherin membrane localization, we generated an mCherry:Numb knockin line and found that Numb localized to diverse endocytic organelles but mainly to the recycling endosome. Consistent with this localization, cardiomyocytes in MDKO did not display defects in N-cadherin internalization but rather in postendocytic recycling to the plasma membrane. Furthermore, N-cadherin overexpression via a mosaic model partially rescued the defects in cellular orientation and trabeculation of MDKO hearts. Our study unravels a phenomenon that cardiomyocytes display spatiotemporal cellular orientation during ventricular wall morphogenesis, and its disruption leads to abnormal trabecular and ventricular wall morphogenesis. Furthermore, we established a mechanism by which Numb modulates cellular orientation and consequently trabecular and ventricular wall morphogenesis by regulating N-cadherin recycling to the plasma membrane.

New insight on the relationship between lethal electrical fields versus cardiomyocyte orientation

Abstract Introduction Cardiovascular diseases represent a major cause of death world-wide and one of their greatest complications is the development of cardiac arrhythmias, in which ventricular fibrillation (VF) stands out as the most severe one. The only therapy that reverses VF is defibrillation. However defibrillatory shock is capable of killing heart cells and it is known that the orientation of the cell major axis with respect to the electrical field (E) direction is a determining factor for cellular excitation and injury, which is leading to the development of new defibrillation protocols. The aim of this work is to fill the gap in information about cell lethality for intermediate cell orientation angles. Methods Ventricular myocytes were extracted from adult male Wistar rats and the cells were plated in a chamber for perfusion and stimulation with bipolar voltage pulses to determine the stimulation threshold (ET). Then, monopolar stimulus was applied and amplitude was increased until cell lethal injury. This protocol was performed on four experimental groups: cells oriented at 0°, 30°, 60° and 90°, with respect to E direction. Results 87 cells were analyzed and an increase in amplitude of E associated with 50% lethality (E50) was verified as the direction of E application and cell major axis orientation departed. Conclusion Taken the same probability of lethality, our data suggest a nonlinear increase of E amplitude from 0° to 90° similar to that of ET. These in-between data had not yet been shown and are important for service-based future defibrillation protocols.

High resolution 3-Dimensional imaging of the human cardiac conduction system from microanatomy to mathematical modeling

Cardiac arrhythmias and conduction disturbances are accompanied by structural remodelling of the specialised cardiomyocytes known collectively as the cardiac conduction system. Here, using contrast enhanced micro-computed tomography, we present, in attitudinally appropriate fashion, the first 3-dimensional representations of the cardiac conduction system within the intact human heart. We show that cardiomyocyte orientation can be extracted from these datasets at spatial resolutions approaching the single cell. These data show that commonly accepted anatomical representations are oversimplified. We have incorporated the high-resolution anatomical data into mathematical simulations of cardiac electrical depolarisation. The data presented should have multidisciplinary impact. Since the rate of depolarisation is dictated by cardiac microstructure, and the precise orientation of the cardiomyocytes, our data should improve the fidelity of mathematical models. By showing the precise 3-dimensional relationships between the cardiac conduction system and surrounding structures, we provide new insights relevant to valvar replacement surgery and ablation therapies. We also offer a practical method for investigation of remodelling in disease, and thus, virtual pathology and archiving. Such data presented as 3D images or 3D printed models, will inform discussions between medical teams and their patients, and aid the education of medical and surgical trainees.

Maturation of human pluripotent stem cell derived cardiomyocytes is improved in cardiovascular construct

In order to translate preclinical data into the clinical studies, relevant in vitro models with structure and key functional properties similar to native human tissue should be used. In vitro cardiac models with vascular structures mimic the highly vascularized myocardium and provide interactions between endothelial cells, stromal cells and cardiomyocytes. Currently, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been shown to present immature morphology and fetal-like electrophysiological properties that may limit their use as physiological test platform. The aim of this study was to develop multicellular in vitro cardiovascular construct modeling human heart tissue. In the cardiovascular construct, hPSC-CMs were cultured with a vascular-like network formed by human foreskin fibroblasts and human umbilical vein endothelial cells that served as a platform in the construct. Cardiomyocyte orientation, maturation, electrophysiological properties and drug responses of the cardiovascular construct were characterized and compared to CM monoculture. hPSC-CMs in cardiovascular construct showed elongated morphology and aligned with the vascular-like network. Electrophysiological properties and calcium metabolism of hPSC-CMs as well as response to E-4031 and adrenaline demonstrated normal physiological behavior. Increased expression of cardiac structural proteins and ion channels in cardiovascular construct compared to CM monoculture were detected. In conclusion, vascular-like network supports the structural and functional maturation of hPSC-CMs. Our results suggest that cardiovascular construct presents more mature in vitro cardiac model compared to CM monoculture and could therefore serve as an advanced test system for cardiac safety and efficacy assessment as well as a model system for biomedical research.

Abstract 20662: Variations in Local Calcium Signaling in Adjacent Cardiomyocytes of the Intact Mouse Heart Detected With Two-Dimensional Confocal Microscopy

Coordinated Ca release in the heart ensures efficient myocardial contraction and electrical stability. Dyssynchronous Ca release within individual cardiomyocytes has been linked to cellular contractile dysfunction. Differences in Ca kinetics in adjacent cells may also provide a substrate for inefficient contraction and arrhythmias. We present an approach to quantify local Ca signals related to the geometry of adjacent cardiomyocytes within the intact heart using 2D confocal microscopy. We observe significant differences in Ca kinetics between adjacent cardiomyocytes. Methods: Ca transients (CaT, Fluo-8 AM) and cell membranes (Di-4 ANEPPS) were visualized in an epicardial area of 465 μm by 315 μm (12 to 20 cells) in Langendorff-perfused mouse hearts. Images (55/second) from a series of CaT at steady state stimulation (250 ms cycle length) were registered to their relative timepoint in the cardiac cycle to restore local CaT with a temporal resolution of 2 ms. Variation of local CaT within a cardiomyocyte (INTRA) and between adjacent cardiomyocytes (INTER) were compared with regard to amplitude, time to peak (TTP) and time constant of decay (TAU). Results: Temporal registration of images from successive CaT yielded local signals with good signal/noise ratio (N= 4 hearts, n= 8 regions). Along the long axis but not along the short axis of cardiomyocyte orientation the variation of CaT amplitude was significantly larger between that within cells (INTER 51.49 ±23.57% vs. INTRA <0.01 ±0.001% of global Ca amplitude). Variation in TTP Ca was comparable in all directions, but TAU also tended to vary more along the long axis (INTER 68.50 ±27.03% vs. INTRA 37.74 ±13.17%). Isoproterenol (10 μM) significantly increased the differences in TAU between adjacent cardiomyocytes along the short axis but not along the long axis. Summary: We present an approach to detect and analyze local Ca signals in Langendorff-perfused intact mouse hearts with high temporal and spatial resolution, taking into account 2D orientation of the cells. We observed significant differences in the synchrony of Ca signals along the long and short axis of adjacent cardioymyocytes. Alterations in synchrony of Ca signals in neighboring cells may contribute to the substrate in cardiac remodeling.

Robust T-tubulation and maturation of cardiomyocytes using tissue-engineered epicardial mimetics

Complex three-dimensional (3-D) heart structure is an important determinant of cardiac electrical and mechanical function. In this study, we set to develop a versatile tissue-engineered system that can promote important aspects of cardiac functional maturation and reproduce variations in myofiber directions present in native ventricular epicardium. We cultured neonatal rat cardiomyocytes within a 3-D hydrogel environment using microfabricated elastomeric molds with hexagonal posts. By varying individual post orientations along the directions derived from diffusion tensor magnetic resonance imaging (DTMRI) maps of human ventricle, we created large (2.5 × 2.5 cm2) 3-D cardiac tissue patches with cardiomyocyte alignment that replicated human epicardial fiber orientations. After 3 weeks of culture, the advanced structural and functional maturation of the engineered 3-D cardiac tissues compared to age-matched 2-D monolayers was evident from: 1) the presence of dense, aligned and electromechanically-coupled cardiomyocytes, quiescent fibroblasts, and interspersed capillary-like structures, 2) action potential propagation with near-adult conduction velocity and directional dependence on local cardiomyocyte orientation, and 3) robust formation of T-tubules aligned with Z-disks, co-localization of L-type Ca2+ channels and ryanodine receptors, and accelerated Ca2+ transient kinetics. This biomimetic tissue-engineered platform can enable systematic in vitro studies of cardiac structure–function relationships and promote the development of advanced tissue engineering strategies for cardiac repair and regeneration.

Electrical Field Stimulation with a Novel Platform: Effect on Cardiomyocyte Gene Expression but not on Orientation

Electrical field stimulation has been shown to improve cardiac cell alignment and functional properties. In this study, neonatal rat cardiomyocytes were exposed to both long-term and short-term stimulation with the goal of investigating whether it is possible to achieve cell orientation and the maturation of cardiomyocytes with a novel, microelectrode array (MEA)-compatible electrical stimulation platform. Cells were viable after electrical stimulation, but no orientation or other morphological changes were observed. However, the electrode wires in MEA dishes affected the cell orientation. Cell contractions synchronized with pacing, but settled back to their original frequency in the absence of stimulation. The expression of genes encoding a gap junction protein connexin-43 (Cx-43), and contractile cardiac protein beta myosin heavy chain 7, was stronger in stimulated cells than in controls (p<0.05). In summary, the surface topography influenced to cardiomyocyte orientation, suggesting that the micro architecture of the biomaterials should be carefully designed for cell applications. However, as electrical stimulation and its duration affected gene expression of some main cardiac proteins, the stimulation system may prove useful to enhance the cardiac differentiation of stem cells.

Progressive changes in T₁, T₂ and left-ventricular histo-architecture in the fixed and embedded rat heart.

Chemical tissue fixation, followed by embedding in either agarose or Fomblin, is common practice in time-intensive MRI studies of ex vivo biological samples, and is required to prevent tissue autolysis and sample motion. However, the combined effect of fixation and sample embedding may alter tissue structure and MRI properties. We investigated the progressive changes in T(1) and T(2) relaxation times, and the arrangement of locally prevailing cardiomyocyte orientation determined using diffusion tensor imaging, in embedded ex vivo rat hearts fixed using Karnovsky's solution (glutaraldehyde-formaldehyde mix). Three embedding media were investigated: (i) standard agarose (n = 3 hearts); (ii) Fomblin (n = 4 hearts); and (iii) iso-osmotic agarose (n = 3 hearts); in the latter, the osmolarity of the fixative and embedding medium was adjusted to 300 mOsm to match more closely that of native tissue. The T(1) relaxation time in the myocardium showed a pronounced decrease over a 48-h period following embedding in Fomblin (-11.3 ± 6.2%; mean ± standard deviation), but was stable in standard agarose- and iso-osmotic agarose-embedded hearts. The mean myocardial T(2) relaxation time increased in all embedded hearts: by 35.1 ± 14.7% with standard agarose embedding, 13.1 ± 5.6% with Fomblin and 13.3 ± 1.4% with iso-osmotic agarose. Deviation in the orientation of the primary eigenvector of the diffusion tensor occurred in all hearts (mean angular changes of 6.6°, 3.2° and 1.9° per voxel after 48 h in agarose-, Fomblin- and iso-osmotic agarose-embedded hearts, respectively), indicative of progressive structural changes in myocardial histo-architecture, in spite of previous exposure to fast-acting tissue fixation. Our results suggest that progressive structural changes occur in chemically fixed myocardium, and that the extent of these changes is modulated by the embedding medium, and by osmotic gradients between the fixative in the tissue and the surrounding medium.

Cyclic Mechanical Stretch Induces Cardiomyocyte Orientation and Polarization of the Gap Junction Protein Connexin43

Cyclic mechanical stretch (CMS) is an important physiological and pathological factor in the heart. We examined whether CMS can affect localization of gap junctions with regard to the cell axis. Neonatal rat cardiomyocytes were cultured (7 days) on flexible 6-well plates. Thereafter, cells were kept static or stimulated with CMS (1 Hz; 0, 10, 20% elongation) for 0, 24, or 48 hours (with or without 10 micromol/L PD98059, 5 micromol/L BIM I (bisindolylmaleimide I), 2 micromol/L H8 [N-(2-methlyamino-ethyl)-5-isoquinoline-sulfonamid], or 0.1 micromol/L angiotensin II. Additionally, cells were exposed to 24 hours of CMS followed by 24 hours of static recovery. CMS (24 hour, 10%) induced elongation of the cardiomyocytes and orientation 79+/-8 degrees toward the stretch direction. Moreover, the distribution of connexin (Cx)43 together with N-cadherin changed, so that both proteins were accentuated at the cell poles, whereas in nonstretched cells, they were distributed around the cell without preferential localization. Additional angiotensin II reduced polar Cx43 accentuation. The CMS-induced changes in Cx43 were reversible within 24 hours after end of stretch, and could be completely prevented by the MEK1/2 inhibitor PD98059 but not by BIM I or H8. Moreover, stretch resulted in Cx43 protein and Cx43-mRNA upregulation and in a significant upregulation of the phosphorylated forms of ERK1/2, glycogen synthase kinase 3beta and AKT. Furthermore, CMS resulted in a significant increase of the transcription factors activator protein 1 and CREB (cAMP response element-binding protein) in the nucleus. CMS results in self-organization of cardiomyocytes leading to elongated cells orientated transverse to the stretch axis, enhanced Cx43 expression and Cx43 accentuation at the cell poles. The Cx43-changes seem to depend on the ERK1/2 signaling cascade.

Interactive effects of surface topography and pulsatile electrical field stimulation on orientation and elongation of fibroblasts and cardiomyocytes

In contractile tissues such as myocardium, functional properties are directly related to the cellular orientation and elongation. Thus, tissue engineering of functional cardiac patches critically depends on our understanding of the interaction between multiple guidance cues such as topographical, adhesive or electrical. The main objective of this study was to determine the interactive effects of contact guidance and electrical field stimulation on elongation and orientation of fibroblasts and cardiomyocytes, major cell populations of the myocardium. Polyvinyl surfaces were abraded using lapping paper with grain size 1–80 μm, resulting in V-shaped abrasions with the average abrasion peak-to-peak width in the range from 3 to 13 μm, and the average depth in the range from 140 to 700 nm (AFM). The surfaces with abrasions 13 μm wide and 700 nm deep, exhibited the strongest effect on neonatal rat cardiomyocyte elongation and orientation as well as statistically significant effect on orientation of fibroblasts, thus they were utilized for electrical field stimulation. Electrical field stimulation was performed using a regime of relevance for heart tissue in vivo as well as for cardiac tissue engineering. Stimulation (square pulses, 1 ms duration, 1 Hz, 2.3 or 4.6 V/cm) was initiated 24 h after cell seeding and maintained for additional 72 h. The cover slips were positioned between the carbon rod electrodes such that the abrasions were either parallel or perpendicular to the field lines. Non-abraded surfaces were utilized as controls. Field stimulation did not affect cell viability. The presence of a well-developed contractile apparatus in neonatal rat cardiomyocytes (staining for cardiac Troponin I and actin filaments) was identified in the groups cultivated on abraded surfaces in the presence of field stimulation. Overall we observed that (i) fibroblast and cardiomyocyte elongation on non-abraded surfaces was significantly enhanced by electrical field stimulation, (ii) electrical field stimulation promoted orientation of fibroblasts in the direction perpendicular to the field lines when the abrasions were also placed perpendicular to the field lines and (iii) topographical cues were a significantly stronger determinant of cardiomyocyte orientation than the electrical field stimulation. The orientation and elongation response of cardiomyocytes was completely abolished by inhibition of actin polymerization (Cytochalasin D) and only partially by inhibition of phosphatidyl-inositol 3 kinase (PI3K) pathway (LY294002).

Tissue Engineering of Vascularized Cardiac Muscle From Human Embryonic Stem Cells

Transplantation of a tissue-engineered heart muscle represents a novel experimental therapeutic paradigm for myocardial diseases. However, this strategy has been hampered by the lack of sources for human cardiomyocytes and by the scarce vasculature in the ischemic area limiting the engraftment and survival of the transplanted muscle. Beyond the necessity of endothelial capillaries for the delivery of oxygen and nutrients to the grafted muscle tissue, interactions between endothelial and cardiomyocyte cells may also play a key role in promoting cell survival and proliferation. In the present study, we describe the formation of synchronously contracting engineered human cardiac tissue derived from human embryonic stem cells containing endothelial vessel networks. The 3D muscle consisted of cardiomyocytes, endothelial cells (ECs), and embryonic fibroblasts (EmFs). The formed vessels were further stabilized by the presence of mural cells originating from the EmFs. The presence of EmFs decreased EC death and increased EC proliferation. Moreover, the presence of endothelial capillaries augmented cardiomyocyte proliferation and did not hamper cardiomyocyte orientation and alignment. Immunostaining, ultrastructural analysis (using transmission electron microscopy), RT-PCR, pharmacological, and confocal laser calcium imaging studies demonstrated the presence of cardiac-specific molecular, ultrastructural, and functional properties of the generated tissue constructs with synchronous activity mediated by action potential propagation through gap junctions. In summary, this is the first report of the construction of 3D vascularized human cardiac tissue that may have unique applications for studies of cardiac development, function, and tissue replacement therapy.

Rac1 activity is required for cardiac myocyte alignment in response to mechanical stress

Mechanical stretch is essential for the cardiac growth. The exposure of cardiac myocytes to the mechanical stretch leads to the cell alignment in parallel to the stretch direction, determining the cell polarity, though it remains to be addressed how mechanical stretch regulates cell orientation. In the present study, we investigated the signal transduction pathways responsible for the cell orientation response to mechanical stretch, focusing on Rho family proteins. Neonatal rat cardiomyocytes were cultured on silicon chambers and exposed to artificial uniaxial cyclic stretch. The pull-down assays revealed that Rac1 was rapidly activated by stretch, but not RhoA. To analyze the roles of Rho family proteins in cardiomyocyte orientation, adenoviral vectors expressing dominant-negative (dn) RhoA and Rac1 were generated. The transfection with adenovirus vector expressing dnRac1, but not dnRhoA, inhibited stretch-induced cell alignment. In conclusion, Rac1 activity is necessary for cardiomyocyte alignment in response to directional stretch.

Control of cardiomyocyte orientation on a microscaffold fabricated by photopolymerization with laser beam interference

We fabricate scaffolds with a fine linear grating (periodicity of 1 to 8 microm and height of 1 microm) on a glass substrate for controlling cardiomyocyte orientation. The fabrication is done by the solidification of a liquid photopolymerizable material using two laser beam interference. As the photopolymerizable material, we use acrylated trimethylene-carbonate-based oligomers initiated with trimethylolpropane (T/TMP), followed by acrylation at terminal ends. Rat cardiomyocytes cultured on the fabricated scaffolds exhibit cell elongation, orientation, and contraction along the scaffold grating. Fluorescence observation of bundles of actin filaments of the cultured cells show that the cytoskeleton of the cells is also generated and oriented parallel to the grating. With a change in grating periodicity from 8 to 1 microm, the percentage of the cells that show orientation along the grating increase from approximately 40 to 70%. The cell orientation along the grating is observed 18 h after seeding the cells on the scaffold. This result implies that the attachment between a cell and a well-defined microarchitectural substrate at an early stage of culture is a significant determinant of cell morphology.